Modulation of stat 6 expression for the treatment of airway hyperresponsiveness

ABSTRACT

Disclosed herein are compounds, compositions and methods for modulating the expression of STAT 6 in a cell, tissue, or animal. Also provided are uses of disclosed compounds and compositions in the manufacture of a medicament for treatment of diseases and disorders related to expression of STAT 6, airway hyperresponsiveness, and/or pulmonary inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application under 35 U.S.C. §371 of International Application No. PCT/US2006/018573, filed May 12,2006, designating the United States and published in English on Nov. 23,2006 as WO2006/124686, which claims priority to U.S. Provisional PatentApplication Ser. No. 60/680,895, filed May 12, 2005 both of which areincorporated herein by reference in their entirety. This application isrelated to US Pregrant Publication Nos. 20040115634 and 20050239124,which are hereby incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The present application is being filed along with a Substitute SequenceListing in electronic format. The Substitute Sequence Listing isprovided as a file entitled BIOL0062USA.txt, created on Nov. 9, 2007which is 112 Kb in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Allergic rhinitis and asthma are widespread conditions with complex andmultifactorial etiologies. The severity of the conditions vary widelybetween individuals, and within individuals, dependent on factors suchas genetics, environmental conditions, and cumulative respiratorypathology associated with duration and severity of disease. Bothdiseases are a result of immune system hyperresponsiveness to innocuousenvironmental antigens, with asthma typically including an atopic (i.e.,allergic) component.

In asthma, the pathology manifests as inflammation, mucusoverproduction, and reversible airway obstruction which may result inscarring and remodeling of the airways. Mild asthma is relatively wellcontrolled with current therapeutic interventions includingbeta-agonists and low dose inhaled corticosteroids or cromolyn. However,moderate and severe asthma are less well controlled, and require dailytreatment with more than one long-term control medication to achieveconsistent control of asthma symptoms and normal lung function. Withmoderate asthma, doses of inhaled corticosteroids are increased relativeto those given to mild asthmatics, and or supplemented with long actingbeta-agonists (LABA) (e.g., salmeterol) or leukotriene inhibitors (e.g.,montelukast, zafirlukast). Although LABA can decrease dependence oncorticosteroids, they are not as effective for total asthma control ascorticosteroids (e.g., reduction of episodes, emergency room visits)(Lazarus et al., JAMA. 2001.285: 2583-2593; Lemanske et al., JAMA. 2001.285: 2594-2603). With severe asthma, doses of inhaled corticosteroidsare increased, and supplemented with both LABA and oral corticosteroids.Severe asthmatics often suffer from chronic symptoms, including nighttime symptoms; limitations on activities; and the need for emergencyroom visits. Additionally, chronic corticosteroid therapy at any levelhas a number of unwanted side effects, especially in children (e.g.,damage to bones resulting in decreased growth).

Allergic rhinitis is inflammation of the nasal passages, and istypically associated with watery nasal discharge, sneezing, congestionand itching of the nose and eyes. It is frequently caused by exposure toirritants, particularly allergens. Allergic rhinitis affects about 20%of the American population and ranks as one of the most common illnessesin the US. Most suffer from seasonal symptoms due to exposure toallergens, such as pollen, that are produced during the natural plantgrowth season(s). A smaller proportion of sufferers have chronicallergies due to allergens that are produced throughout the year such ashouse dust mites or animal dander. A number of over the countertreatments are available for the treatment of allergic rhinitisincluding oral and nasal antihistamines, and decongestants.Antihistamines are utilized to block itching and sneezing and many ofthese drugs are associated with side effects such as sedation andperformance impairment at high doses. Decongestants frequently causeinsomnia, tremor, tachycardia, and hypertension. Nasal formulations,when taken improperly or terminated rapidly, can cause reboundcongestion. Anticholinergics and montelukast have substantially fewerside effects, but they also have limited efficacy. Similarly,prescription medications are not free of side effects. Nasalcorticosteroids can be used for prophylaxis or suppression of symptoms;however, compliance is variable due to side effects including poor tasteand nasal irritation and bleeding. Allergen immunotherapy is expensiveand time consuming and carries a low risk of anaphylaxis.

Persistent nasal inflammation can result in the development of nasalpolyps. Nasal polyps are present in about 4.2% of patients with chronicrhinitis and asthma (4.4% of men and 3.8% of women) (Grigores et al.,Allergy Asthma Proc. 2002, 23:169-174). The presence of polyps isincreased with age in both sexes and in patients with cystic fibrosisand aspirin-hypersensitivity triad. Nasal polyposis results from chronicinflammation of the nasal and sinus mucous membranes. Chronicinflammation causes a reactive hyperplasia of the intranasal mucosalmembrane, which results in the formation of polyps. The precisemechanism of polyp formation is incompletely understood. Nasal polypsare associated with nasal airway obstruction, postnasal drainage, dullheadaches, snoring, anosmia, and rlinorrhea. Medical therapies includetreatment for underlying chronic allergic rhinitis using antihistaminesand topical nasal steroid sprays. For severe nasal polyposis causingsevere nasal obstruction, treatment with short-term steroids may bebeneficial. Topical use of cromolyn spray has also been found to behelpful to some patients in reducing the severity and size of the nasalpolyps. Oral corticosteroids are the most effective medication for theshort-term treatment of nasal polyps, and oral corticosteroids have thebest effectiveness in shrinking inflammatory polyps. Intranasal steroidsprays may reduce or retard the growth of small nasal polyps, but theyare relatively ineffective in massive nasal polyposis. Although nasalpolyps can be treated pharmacologically, many of the therapeutics haveundesirable side effects. Moreover, polyps tend to be recurrent,eventually requiring surgical intervention. Compositions and methods toinhibit post-surgical recurrence of nasal polyps are not presentlyavailable.

Other diseases characterized by similar inflammatory pathways include,but are not limited to, chronic bronchitis, pulmonary fibrosis,emphysema, chronic obstructive pulmonary disease (COPD), eosinophilicpneumonia, and pediatric asthma.

Signal Transducer and Activator of Transcription 6 (STAT 6) andInflammatory Signaling Pathways

It is generally acknowledged that allergy and asthma are a result of thedysregulation of T cell-mediated immunity resulting in a bias towards aTh2 response (enhanced production of interleukin-4 (IL-4) IL-5 andIL-13). The presence of CD4+ T cells producing IL-4, IL-5 and IL-13cytokines in bronchioalveolar lavage fluid and in airway epithelialbiopsies of asthmatics has been clearly documented. STAT 6 is anintegral transcription factor involved in interleukin 4 and interleukin13 signaling. Following activation of their respective receptors,interleukin 4 and interleukin 13 cause their common interleukin 4receptor alpha chain to become phosphorylated by JAK3 and tosubsequently bind to STAT 6. STAT 6 is then phosphorylated by JAK1,homodimerizes and translocates to the nucleus where it binds interleukin4 response elements and initiates the transcription of a number of genesincluding IgE (Danahay et al., Inflamm. Res., 2000, 49, 692-699).

STAT 6 (also known as interleukin 4-STAT) was cloned and mapped tochromosome 12q13 (Leek et al., Cytogenet. Cell Genet., 1997, 79,208-209; Quelle et al., Mol. Cell. Biol., 1995, 15, 3336-3343). Nucleicacid sequences encoding STAT 6 are disclosed and claimed in U.S. Pat.No. 5,710,266 (McKnight and Hou, 1998).

STAT 6 is primarily expressed as a 4 kb transcript in hematopoieticcells and expressed variably in other tissues (Quelle et al., Mol. Cell.Biol., 1995, 15, 3336-3343). A unique truncated isoform of STAT 6 isexpressed in mast cells (Sherman, Immunol. Rev., 2001, 179, 48-56).Disclosed and claimed in PCT publication WO 99/10493 are nucleic acidsequences encoding variants of STAT 6 known as STAT 6b and STAT 6c aswell as vectors comprising said nucleic acid sequences (Patel et al.,1999).

STAT 6 knockout mice are viable and develop normally with the exceptionthat interleukin 4 functions are eliminated (Ihle, Curr. Opin. CellBiol., 2001, 13, 211-217). Additionally, STAT 6 knockout mice fail todevelop antigen-induced airway hyper-reactivity in a model of airwayinflammation (Kuperman et al., J. Exp. Med., 1998, 187, 939-948).

Inhibition of STAT 6 is expected to attenuate the allergic response andthus, represents an attractive target for drug discovery strategies(Hill et al., Am. J. Respir. Cell Mol. Biol., 1999, 21, 728-737).

Small molecule inhibitors of STAT 6 are disclosed and claimed in PCTpublication WO 00/27802 and Japanese Patent JP 2000229959 (Eyermann etal., 2000; Inoue et al., PCT, 2000, Abstract only). Disclosed andclaimed in U.S. Pat. No. 6,207,391 are methods for screening modulatorsof STAT 6 binding to a STAT 6 receptor (Wu and McKinney, 2001).

Wang et al. have demonstrated targeted disruption of STAT 6 DNA-bindingactivity by a phosphorothioate cis-element decoy oligonicleotide (Wanget al., Blood, 2000, 95, 1249-1257). Hill et al. have used a series ofhomologous human and murine antisense oligonucleotides targeting STAT 6to interrupt interleukin 4 and interleukin 13 signaling and attenuategermline C-epsilon transcription in vitro (Hill et al., Am. J. Respir.Cell Mol. Biol., 1999, 21, 728-737). Subsequently, the in vitro and invivo pharmacology of three of the antisense oligonucleotides used in thelatter study was investigated. Although the oligonucleotidesdownregulated STAT 6 mRNA, their action was not sufficient to influencealterations in IgE levels in a model of active sensitization (Danahay etal., Inflamm. Res., 2000, 49, 692-699). Although the oligonucleotideswere able to decrease target expression in the spleen, splenomegaly wasobserved, indicating immune-stimulation by the oligonucleotides. USPregrant Publications Nos. 20040115634 and 20050239124 teach a series ofoligonucleotides targeted to STAT 6.

Antisense Oligonucleotides and Pulmonary Disease

Antisense oligonucleotides (ASOs) are being pursued as therapeutics forpulmonary inflammation, airway hyperresponsiveness, and/or asthma. Lungprovides an ideal tissue for aerosolized ASOs for several reasons (Nyceand Metzger, Nature, 1997: 385:721-725, incorporated herein by referencein its entirety); the lung can be targeted non-invasively andspecifically, it has a large absorption surface; and is lined withsurfactant that may facilitate distribution and uptake of ASOs. Deliveryof ASOs to the lung by aerosol results in excellent distributionthroughout the lung in both mice and primates. Immunohistochemicalstaining of inhaled ASOs in normalized and inflamed mouse lung tissueshows heavy staining in alveolar macrophages, eosinophils, andepithelium, moderate staining in blood vessels endothelium, and weakstaining in bronchiolar epithelium. ASO— mediated target reduction isobserved in dendritic cells, macrophages, eosinophils, and epithelialcells after aerosol administration. The estimated half life of a2′-methoxyethoxy (2′-MOE) modified oligonucleotide delivered by aerosoladministration to mouse or monkey is about 4 to 7, or at least 7 days,respectively. Moreover, ASOs have relatively predictable toxicities andpharmacokinetics based on backbone and nucleotide chemistry. Pulmonaryadministration of ASOs results in minimal systemic exposure, potentiallyincreasing the safety of such compounds as compared to other classes ofdrugs.

Compositions and methods for formulation of ASOs and devices fordelivery to the lung and nose are well known. ASOs are soluble inaqueous solution and may be delivered using standard nebulizer devices(Nyce, Exp. Opin. Invest. Drugs, 1997, 6:1149-1156). Formulations andmethods for modulating the size of droplets using nebulizer devices totarget specific portions of the respiratory tract and lungs are wellknown to those skilled in the art. Oligonucleotides can be deliveredusing other devices such as dry powder inhalers or metered dose inhalerswhich can provide improved patient convenience as compared to nebulizerdevices, resulting in greater patient compliance.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and effects themodulation of gene expression activity, or function, such astranscription or translation. The modulation of gene expression can beachieved by, for example, target RNA degradation or occupancy-basedinhibition. An example of modulation of target RNA function bydegradation is RNase H-based degradation of the target RNA uponhybridization with a DNA-like antisense compound. Another example ofmodulation of gene expression by target degradation is RNA interference(RNAi) using small interfering RNAs (siRNAs). RNAi is a form ofantisense-mediated gene silencing involving the introduction of doublestranded (ds)RNA-like oligonucleotides leading to the sequence-specificreduction of targeted endogenous mRNA levels. This sequence-specificitymakes antisense compounds extremely attractive as tools for targetvalidation and analysis of gene function, as well as therapeutics toselectively modulate the expression of genes involved in diseases.

Antisense oligonucleotides targeted to a number of targets including,but not limited to p38 alpha MAP kinase (US Patent Publication No.20040171566, incorporated by reference); the CD28 receptor ligands B7.1and B7.2 (US Patent Publication 20040235164, incorporated by reference);intracellular adhesion molecule (ICAM) (WO 2004/108945, incorporated byreference); and adenosine A₁ receptor (Nyce and Metzger, Nature, 1997,385:721-725, incorporated herein by reference) have been tested fortheir ability to inhibit pulmonary inflammation and airwayhyperresponsiveness in mouse, rabbit, and/or monkey models of asthmawhen delivered by inhalation. Various endpoints were analyzed in eachcase and a portion of the results are presented herein. ASOs targeted top38 alpha MAP kinase reduced eosinophil recruitment, airwayhyperresponsiveness (AHR), and mucus production in two different mousemodels. ASOs targeted to each B7.1 and B7.2 decreased target expressionand eosinophil recruitment. An ASO targeted to B7.2 also reduced AHR.ASOs targeted to ICAM-1 decreased AHR and decreased neutrophil andeosinophil recruitment in mice. Treatment of Cynomolgus monkeys with anASO targeted to ICAM-1 significantly reduced airway impedance(resistance) induced by methacholine challenge in naturally Ascarisallergen-sensitized monkeys. An ASO targeted to adenosine A₁ receptorreduced receptor density on airway smooth muscle and reduced AHR in anallergic rabbit model. These data demonstrate that oligonucleotides areeffectively delivered by inhalation to cells within the lungs ofmultiple species, including a non-human primate, and are effective atreducing airway hyperresponsiveness and/or pulmonary inflammation asdetermined by a number of endpoints.

However, treatment with any ASO targeted to any inflammatory mediatorinvolved in pulmonary inflammation is not always effective at reducingAHR and/or pulmonary inflammation. ASOs targeted to Jun N-terminalKinase (JNK-1) found to decrease target expression in vitro were testedin a mouse model of asthma. Treatment with each of two differentantisense oligonucleotides targeted to JNK-1 were not effective atreducing methacholine induced AHR, eosinophil recruitment, or mucusproduction at any of the ASO doses tested.

A number of ASOs designed to target STAT 6 have been reported for use asresearch tools. The PCT publication WO02088328 (Belardelli et al., 2002)discloses the use of an oligonucleotide of 24 nucleotides in length thatis complementary to a nucleic acid molecule encoding STAT 6. U.S. Pat.No. 6,699,677 (Schall et al., 2004) discloses the use of anoligonucleotide of 30 nucleotides in length as a PCR primer foramplifying a nucleic acid molecule encoding STAT 6. The PCT publicationWO0240647 (Ulrich and Saikh, 2002) discloses the use of anoligonucleotide of 30 nucleotides in length as a PCR primer foramplifying a nucleic acid molecule encoding STAT 6. A series ofantisense oliognucleotides targeted to STAT 6 are taught in US PatentPublication US2004-0115634.

The role of STAT 6 in the Th2 inflammatory signaling pathways makes itan attractive therapeutic candidate, as this pathway has been linked toasthma, allergy, and other inflammatory disorders. Currently, there areno known therapeutic agents that effectively inhibit the synthesis ofSTAT 6, and all investigative strategies to date aimed at modulatingfunction have involved the use of antibodies. Consequently, thereremains a need for additional agents capable of effectively inhibitingthe activity of STAT 6.

SUMMARY OF THE INVENTION

The invention provides compounds, particularly antisense compounds,especially nucleic acid and nucleic acid-like oligomers, which aretargeted to a nucleic acid encoding STAT 6. Preferably, the antisensecompounds are antisense oligonucleotides targeted to STAT 6,particularly human STAT 6 (GenBank Accession No. NM_(—)003153.3, enteredOct. 1, 2002 (SEQ ID NO. 1)), that modulate the expression of STAT 6.The compounds comprise at least a 12 nucleobase portion, preferably atleast a 15 nucleobase portion, most preferably at least a 17 nucleobaseportion targeted to an active target segment, or are at least 80%complementary to at least a 15 nucleobase portion an active targetsegments.

The invention provides a method for modulating the expression of STAT 6in cells or tissues comprising contacting the cells with at least onecompound of the instant invention, and analyzing the cells forindicators of a decrease in expression of STAT 6 mRNA and/or protein bydirect measurement of mRNA and/or protein levels, and/or indicators ofpulmonary inflammation and/or airway hyperresponsiveness.

The invention further provides a method for the prevention,amelioration, and/or treatment of pulmonary inflammation and/or airwayhyperresponsiveness comprising administering at least one compound ofthe instant invention to an individual in need of such intervention. Thecompound is preferably administered by aerosol (i.e., topically) to atleast a portion of the respiratory tract. The portion of the respiratorytract selected is dependent upon the location of the inflammation. Forexample, in the case of asthma, the compound is preferably deliveredpredominantly to the lung. In the case of allergic rhinitis, thecompound is preferably delivered predominantly to the nasal cavityand/or sinus. The compound is delivered using any of a number ofstandard delivery devices and methods well known to those skilled in theart, including, but not limited to nebulizers, nasal and pulmonaryinhalers, dry powder inhalers, and metered dose inhalers.

The invention also provides a method of use of the compositions of theinstant invention for the preparation of a medicament for theprevention, amelioration, and/or treatment disease, especially a diseaseassociated with and including at least one indicator of pulmonaryinflammation and/or airway hyperresponsiveness. The medicament ispreferably formulated for aerosol administration to at least a portionof the respiratory tract.

DETAILED DESCRIPTION OF THE INVENTION

Asthma, allergy, and a number of other diseases or conditions related topulmonary inflammation and/or AHR share common inflammatory mediators,including STAT 6, a Th2 cytokine. Therapeutic interventions for thesediseases or conditions are not completely satisfactory due to lack ofefficacy and/or unwanted side effects of the compounds. The instantinvention provides antisense compounds, preferably antisense compounds,for the prevention, amelioration, and/or treatment of pulmonaryinflammation and/or airway hyperresponsiveness. As used herein, the term“prevention” means to delay or forestall onset or development of acondition or disease for a period of time from hours to days, preferablyweeks to months. As used herein, the term “amelioration” means alessening of at least one indicator of the severity of a condition ordisease. The severity of indicators may be determined by subjective orobjective measures which are known to those skilled in the art. As usedherein, “treatment” means to administer a composition of the inventionto effect an alteration or improvement of the disease or condition.Prevention, amelioration, and/or treatment may require administration ofmultiple doses at regular intervals, or prior to exposure to an agent(e.g., an allergen) to alter the course of the condition or disease.Moreover, a single agent may be used in a single individual for eachprevention, amelioration, and treatment of a condition or disease,sequentially or concurrently. In a preferred method of the instantinvention, the ASOs are delivered by aerosol for topical delivery to therespiratory tract, thereby limiting systemic exposure and reducingpotential side effects.

Overview

Disclosed herein are antisense compounds, including antisenseoligonucleotides and other antisense compounds for use in modulating theexpression of nucleic acid molecules encoding STAT 6. This isaccomplished by providing antisense compounds that hybridize with one ormore target nucleic acid molecules encoding STAT 6. As used herein, theterms “target nucleic acid” and “nucleic acid molecule encoding STAT 6”have been used for convenience to encompass RNA (including pre-mRNA andmRNA or portions thereof) transcribed from DNA encoding STAT 6, and alsocDNA derived from such RNA. In a preferred embodiment, the targetnucleic acid is an mRNA encoding STAT 6.

The principle behind antisense technology is that an antisense compoundhybridizes to a target nucleic acid to modulate gene expressionactivities such as transcription or translation. This sequencespecificity makes antisense compounds extremely attractive fortherapeutics to selectively modulate the expression of genes involved indisease, as well as tools for target validation and gene functionalanalysis. Although not limited by mechanism of action, the compounds ofthe instant invention are proposed to work by an antisense,non-autocatalytic mechanism.

Target Nucleic Acids

“Targeting” an antisense compound to a particular target nucleic acidmolecule can be a multistep process. The process usually begins with theidentification of a target nucleic acid whose expression is to bemodulated. For example, the target nucleic acid can be a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. As disclosed herein the target nucleic acid encodesSTAT 6.

Variants

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants.” More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence. Variants can result in mRNA variants including, but notlimited to, those with alternate splice junctions, or alternateinitiation and termination codons. Variants in genomic and mRNAsequences can result in disease. Antisense compounds targeted to suchvariants are within the scope of the instant invention.

Target Names, Synonyms, Features

In accordance with the present invention are compositions and methodsfor modulating the expression of STAT 6 (also known as IL4-STAT). Thereare also a number of isoforms of STAT 6 including STAT 6a (the mainmRNA), STAT 6b, STAT 6c, STAT 6d, and STAT 6e. Table 1 lists the GenBankaccession numbers of sequences corresponding to nucleic acid moleculesencoding STAT 6 (nt=nucleotide), the date the version of the sequencewas entered in GenBank, the isoform if not representing the main mRNA,and the corresponding SEQ ID NO in the instant application, whenassigned, each of which is incorporated herein by reference. TABLE 1Gene Targets STAT 6 SEQ Species Genbank # GenBank Date Isoform ID NOHuman NM_003153.1 Mar. 24, 1999 4 Human BC005823.1 Apr. 4, 2001 5 HumanAC018673.4, nt 157501- Nov. 30, 2000 6 174000 Human BE972840.1 Oct. 4,2000 d 7 Human BF902909.1 Jan. 18, 2001 e 8 Human NM_003153.3 Oct. 1,2002 1 Human AR204914.1 Jun. 20, 2002 b 9 Human AR204915.1 Jun. 20, 2002c 10 Human AF067572.1 Oct. 25, 1998 11 Human AF067573.1 Oct. 25, 1998 12Human AF067574.1 Oct. 25, 1998 13 Human AF067575.1 Oct. 25, 1998 14Mouse NM_009284.1 Jan. 6, 2000 15 Mouse BY723237.1 Dec. 17, 2002 16Mouse BC029318.1 Nov. 19, 2003 17Modulation of Target Expression

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid (DNA or RNA) functions.“Modulation” means a perturbation of function, for example, either anincrease (stimulation or induction) or a decrease (inhibition orreduction) in expression. As another example, modulation of expressioncan include perturbing splice site selection of pre-mRNA processing.“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. These structures include the products of transcription andtranslation. “Modulation of expression” means the perturbation of suchfunctions. The functions of RNA to be modulated can includetranslocation functions, which include, but are not limited to,translocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, and translation of protein from the RNA. RNA processingfunctions that can be modulated include, but are not limited to,splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.One result of such interference with target nucleic acid function ismodulation of the expression of STAT 6. Thus, in one embodimentmodulation of expression can mean increase or decrease in target RNA orprotein levels. In another embodiment modulation of expression can meana decrease or increase of one or more RNA splice products, or a changein the ratio of two or more splice products.

The effect of antisense compounds of the present invention on targetnucleic acid expression can be tested in any of a variety of cell typesprovided that the target nucleic acid is present at measurable levels.The effect of antisense compounds of the present invention on targetnucleic acid expression can be routinely determined using, for example,PCR or Northern blot analysis. Cell lines are derived from both normaltissues and cell types and from cells associated with various disorders(e.g. hyperproliferative disorders). Cell lines derived from multipletissues and species can be obtained from American Type CultureCollection (ATCC, Manassas, Va.) and other public sources, and are wellknown to those skilled in the art. Primary cells, or those cells whichare isolated from an animal and not subjected to continuous culture, canbe prepared according to methods known in the art, or obtained fromvarious commercial suppliers. Additionally, primary cells include thoseobtained from donor human subjects in a clinical setting (i.e. blooddonors, surgical patients). Primary cells prepared by methods known inthe art.

Assaying Modulation of Expression

Modulation of STAT 6 expression can be assayed in a variety of waysknown in the art. STAT 6 mRNA levels can be quantitated by, e.g.,Northern blot analysis, competitive polymerase chain reaction (PCR), orreal-time PCR. RNA analysis can be performed on total cellular RNA orpoly(A)+ mRNA by methods known in the art. Methods of RNA isolation aretaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley& Sons, Inc., 1993.

Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. The method of analysis ofmodtdation of RNA levels is not a limitation of the instant invention.

Levels of a protein encoded by STAT 6 can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to a protein encoded by STAT 6 can beidentified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional antibody generation methods. Methods forpreparation of polyclonal antisera are taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997.

Active Target Segments

The locations on the target nucleic acid defined by having one or moreactive antisense compounds targeted thereto are referred to as “activetarget segments.” When an active target segment is defined by multipleantisense compounds, the compounds are preferably separated by no morethan about 50 nucleotides on the target sequence, more preferably nomore than about 10 nucleotides on the target sequence, even morepreferably the compounds are contiguous, most preferably the compoundsare overlapping. There may be substantial variation in activity (e.g.,as defined by percent inhibition) of the antisense compounds within anactive target segment. Active antisense compounds are those thatmodulate the expression of their target RNA in the methods describedherein. Active antisense compounds inhibit expression of their targetRNA at least about 50%. In a preferred embodiment, at least about 50%,of the oligonucleotides targeted to the active target segment modulateexpression of their target RNA at least 65%. In a more preferredembodiment, the level of inhibition required to define an activeantisense compound is defined based on the results from the screen usedto define the active target segments.

Hybridization

As used herein, “hybridization” means the pairing of complementarystrands of antisense compounds to their target sequence. While notlimited to a particular mechanism, the most common mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleobases). For example, the natural base adenine iscomplementary to the natural nucleobases thymidine and uracil which pairthrough the formation of hydrogen bonds. The natural base guanine iscomplementary to the natural bases cytosine and 5-methyl cytosine.Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

As used herein, “stringent hybridization conditions” or “stringentconditions” refers to conditions under which an antisense compound willhybridize to its target sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances, and “stringent conditions” underwhich antisense compounds hybridize to a target sequence are determinedby the nature and composition of the antisense compounds and the assaysin which they are being investigated.

Complementarity

“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases on either two oligomeric compoundstrands or an antisense compound with its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position.

“Complementarity” can also be viewed in the context of an antisensecompound and its target, rather than in a base by base manner. Theantisense compound and the further DNA or RNA are complementary to eachother when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the antisense compound and a targetnucleic acid. One skilled in the art recognizes that the inclusion ofmismatches is possible without eliminating the activity of the antisensecompound. The invention is therefore directed to those antisensecompounds that may contain up to about 20% nucleotides that disrupt basepairing of the antisense compound to the target. Preferably thecompounds contain no more than about 15%, more preferably not more thanabout 10%, most preferably not more than 5% or no mismatches. Theremaining nucleotides do not disrupt hybridization (e.g., universalbases).

Identity

Antisense compounds, or a portion thereof, may have a defined percentidentity to a SEQ ID NO, or a compound having a specific Isis number. Asused herein, a sequence is identical to the sequence disclosed herein ifit has the same nucleobase pairing ability. For example, a RNA whichcontains uracil in place of thymidine in the disclosed sequences of theinstant invention would be considered identical as they both pair withadenine. This identity may be over the entire length of the oligomericcompound, or in a portion of the antisense compound (e.g., nucleobases1-20 of a 27-mer may be compared to a 20-mer to determine percentidentity of the oligomeric compound to the SEQ ID NO.) It is understoodby those skilled in the art that an antisense compound need not have anidentical sequence to those described herein to function similarly tothe antisense compound described herein. Shortened versions of antisensecompound taught herein, or non-identical versions of the antisensecompound taught herein fall within the scope of the invention.Non-identical versions are those wherein each base does not have thesame pairing activity as the antisense compounds disclosed herein. Basesdo not have the same pairing activity by being shorter or having atleast one abasic site. Alternatively, a non-identical version caninclude at least one base replaced with a different base with differentpairing activity (e.g., G can be replaced by C, A, or T). Percentidentity is calculated according to the number of bases that haveidentical base pairing corresponding to the SEQ ID NO or antisensecompound to which it is being compared. The non-identical bases may beadjacent to each other, dispersed through out the oligonucleotide, orboth.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a20-mer is 80% identical to the 20-mer. Alternatively, a 20-mercontaining four nucleobases not identical to the 20-mer is also 80%identical to the 20-mer. A 14-mer having the same sequence asnucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Suchcalculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in theoriginal sequence present in a portion of the modified sequence.Therefore, a 30 nucleobase antisense compound comprising the fullsequence of the complement of a 20 nucleobase active target segmentwould have a portion of 100% identity with the complement of the 20nucleobase active target segment, while further comprising an additional10 nucleobase portion. In the context of the invention, the complementof an active target segment may constitute a single portion. In apreferred embodiment, the oligonucleotides of the instant invention areat least about 80%, more preferably at least about 85%, even morepreferably at least about 90%, most preferably at least 95% identical toat least a portion of the complement of the active target segmentspresented herein.

It is well known by those skilled in the art that it is possible toincrease or decrease the length of an antisense compound and/orintroduce mismatch bases without eliminating activity. For example, inWoolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992,incorporated herein by reference), a series of ASOs 13-25 nucleobases inlength were tested for their ability to induce cleavage of a target RNA.ASOs 25 nucleobases in length with 8 or 11 mismatch bases near the endsof the ASOs were able to direct specific cleavage of the target mRNA,albeit to a lesser extent than the ASOs that contained no mismatches.Similarly, target specific cleavage was achieved using a 13 nucleobaseASOs, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc.Acid. Res. 16:3341-3358, 1988, incorporated herein by reference) testeda series of tandem 14 nucleobase ASOs, and a 28 and 42 nucleobase ASOscomprised of the sequence of two or three of the tandem ASOs,respectively, for their ability to arrest translation of human DHFR in arabbit reticulocyte assay. Each of the three 14 nucleobase ASOs alonewere able to inhibit translation, albeit at a more modest level than the28 or 42 nucleobase ASOs. It is understood that antisense compounds ofthe instant invention can vary in length and percent complementarity tothe target provided that they maintain the desired activity. Methods todetermine desired activity are disclosed herein and well known to thoseskilled in the art.

Therapeutics

Antisense compounds of the invention can be used to modulate theexpression of STAT 6 in an animal, such as a human. In one non-limitingembodiment, the methods comprise the step of administering to saidanimal in need of therapy for a disease or condition associated withSTAT 6 an effective amount of an antisense compound that inhibitsexpression of STAT 6. A disease or condition associated with STAT 6includes, but is not limited to, pulmonary inflammation and airwayhyperresponsiveness. In one embodiment, the antisense compounds of thepresent invention effectively inhibit the levels or function of STAT 6RNA. Because reduction in STAT 6 mRNA levels can lead to alteration inSTAT 6 protein products of expression as well, such resultantalterations can also be measured. Antisense compounds of the presentinvention that effectively inhibit the level or function of STAT 6 RNAor protein products of expression are considered active antisensecompounds. In one embodiment, the antisense compounds of the inventioninhibit the expression of STAT 6 causing a reduction of RNA, preferablyin target cells or tissues, by at least 10%, by at least 20%, by atleast 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99%, or by 100%.

For example, the reduction of the expression of STAT 6 can be measuredin a bodily fluid, which may or may not contain cells; tissue, or organof the animal. Methods of obtaining samples for analysis, such as bodyfluids (e.g., sputum, serum), tissues (e.g., biopsy), or organs, andmethods of preparation of the samples to allow for analysis are wellknown to those skilled in the art. Methods for analysis of RNA andprotein levels are discussed above and are well known to those skilledin the art. The effects of treatment can be assessed by measuringbiomarkers associated with the target gene expression in theaforementioned fluids, tissues or organs, collected from an animalcontacted with one or more compounds of the invention, by routineclinical methods known in the art. These biomarkers include but are notlimited to: liver transaminases, bilirubin, albumin, blood tureanitrogen, creatine and other markers of kidney and liver function;interleukins, tumor necrosis factors, intracellular adhesion molecules,C-reactive protein chemokines, cytokines, and other markers ofinflammation.

The antisense compounds of the present invention can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Acceptablecarriers and dilutents are well known to those skilled in the art.Selection of a dilutent or carrier is based on a number of factors,including, but not limited to, the solubility of the compound and theroute of administration. Such considerations are well understood bythose skilled in the art. In one aspect, the antisense compounds of thepresent invention inhibit the expression of STAT 6. The compounds of theinvention can also be used in the manufacture of a medicament for thetreatment of diseases and disorders related to STAT 6 expression.

Methods whereby bodily fluids, organs or tissues are contacted with aneffective amount of one or more of the antisense compounds orcompositions of the invention are also contemplated. Bodily fluids,organs or tissues can be contacted with one or more of the compounds ofthe invention resulting in modulation of STAT 6 expression in the cellsof bodily fluids, organs or tissues. An effective amount can bedetermined by monitoring the modulatory effect of the antisense compoundor compounds or compositions on target nucleic acids or their productsby methods routine to the skilled artisan.

Thus, provided herein is the use of an isolated single- ordouble-stranded antisense compound targeted to STAT 6 in the manufactureof a medicament for the treatment of a disease or disorder by means ofthe method described above. In a preferred embodiment, the antisensecompound is a single stranded antisense compound. Such antisensecompounds can function by any of a number of non-autocatalyticmechanisms including by the action of RNases (e.g., RNaseH) ormodulation of splicing. Alternative antisense mechanisms (e.g., RNAi)can be promoted by the inclusion of a second, complementary strand tothe antisense compound and/or inclusion of specific chemicalmodifications which are known to those skilled in the art.

Kits, Research Reagents, and Diagnostics

The antisense compounds of the present invention can be utilized fordiagnostics, and as research reagents and kits. Furthermore, antisensecompounds, which are able to inhibit gene expression with specificity,are often used by those of ordinary skill to elucidate the function ofparticular genes or to distinguish between functions of various membersof a biological pathway.

For use in kits and diagnostics, the antisense compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues. Methods of geneexpression analysis are well known to those skilled in the art.

Compounds

The term “oligomeric compound” refers to a polymeric structure capableof hybridizing to a region of a nucleic acid molecule. Generally,oligomeric compounds comprise a plurality of monomeric subunits linkedtogether by internucleoside linking groups and/or internucleosidelinkage mimetics. Each of the monomeric subunits comprises a sugar,abasic sugar, modified sugar, or a sugar mimetic, and except for theabasic sugar includes a nucleobase, modified nucleobase or a nucleobasemimetic. Preferred monomeric subunits comprise nucleosides and modifiednucleosides.

An “antisense compound” or “antisense oligomeric compound” refers to anoligomeric compound that is at least partially complementary to theregion of a target nucleic acid molecule to which it hybridizes andwhich modulates (increases or decreases) its expression. This termincludes oligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics, antisense compounds, antisense oligomericcompounds, and chimeric combinations of these. Consequently, while allantisense compounds can be said to be oligomeric compounds, not alloligomeric compounds are antisense compounds. An “antisenseoligonucleotide” is an antisense compound that is a nucleic acid-basedoligomer. An antisense oligonucleotide can, in some cases, include oneor more chemical modifications to the sugar, base, and/orinternucleoside linkages. Nonlimiting examples of antisense compoundsinclude primers, probes, antisense compounds, antisenseoligonucleotides, external guide sequence (EGS) oligonucleotides,alternate splicers, and siRNAs. As such, these compounds can beintroduced in the form of single-stranded, double-stranded, circular,branched or hairpins and can contain structural elements such asinternal or terminal bulges or loops. Antisense double-strandedcompounds can be two strands hybridized to form double-strandedcompounds or a single strand with sufficient self complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The compounds of the instant invention are notauto-catalytic. As used herein, “auto-catalytic” means a compound hasthe ability to promote cleavage of the target RNA in the absence ofaccessory factors, e.g. proteins.

In one embodiment of the invention, the antisense compound comprises asingle stranded oligonucleotide. In some embodiments of the inventionthe antisense compound contains chemical modifications. In a preferredembodiment, the antisense compound is a single stranded, chimericoligonucleotide wherein the modifications of sugars, bases, andinternucleoside linkages are independently selected.

The antisense compounds in accordance with this invention may comprisean antisense compound from about 12 to about 35 nucleobases (i.e. fromabout 12 to about 35 linked nucleosides). In other words, asingle-stranded compound of the invention comprises from about 12 toabout 35 nucleobases, and a double-stranded antisense compound of theinvention (such as a siRNA, for example) comprises two strands, each ofwhich is independently from about 12 to about 35 nucleobases. Thisincludes oligonucleotides 15 to 35 and 16 to 35 nucleobases in length.Contained within the antisense compounds of the invention (whethersingle or double stranded and on at least one strand) are antisenseportions. The “antisense portion” is that part of the antisense compoundthat is designed to work by one of the aforementioned antisensemechanisms. One of ordinary skill in the art will appreciate that about12 to about 35 nucleobases includes 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35nucleobases.

Antisense compounds about 12 to 35 nucleobases in length, preferablyabout 15 to 35 nucleobases in length, comprising a stretch of at leasteight (8), preferably at least 12, more preferably at least 15consecutive nucleobases targeted to the active target regions areconsidered to be suitable antisense compounds as well.

Modifications can be made to the antisense compounds of the instantinvention and may include conjugate groups attached to one of thetermini, selected nucleobase positions, sugar positions or to one of theinternucleoside linkages. Possible modifications include, but are notlimited to, 2′-fluoro (2′-F), 2′-OMethyl (2′-OMe), 2′-Methoxy ethoxy(2′-MOE) sugar modifications, inverted abasic caps, deoxynucleobases,and bicyclice nucleobase analogs such as locked nucleic acids (includingLNA) and ENA.

In one embodiment of the invention, double-stranded antisense compoundsencompass short interfering RNAs (siRNAs). As used herein, the term“siRNA” is defined as a double-stranded compound having a first andsecond strand, each strand having a central portion and two independentterminal portions. The central portion of the first strand iscomplementary to the central portion of the second strand, allowinghybridization of the strands. The terminal portions are independently,optionally complementary to the corresponding terminal portion of thecomplementary strand. The ends of the strands may be modified by theaddition of one or more natural or modified nucleobases to form anoverhang.

Each strand of the siRNA duplex may be from about 12 to about 35nucleobases. In a preferred embodiment, each strand of the siRNA duplexis about 17 to about 25 nucleobases. The two strands may be fullycomplementary (i.e., form a blunt ended compound), or include a 5′ or 3′overhang on one or both strands. Double-stranded compounds can be madeto include chemical modifications as discussed herein. Structures ofsiRNAs are well known to those skilled in the art (see e.g., Guo andKempheus, Cell, 1995, 81, 611-620; Montgomery et al., Proc. Natl. Acad.Sci. USA, 1998, 95, 15502-15507; and Fire et al., Nature, 1998, 391,806-811).

Chemical Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base(sometimes referred to as a “nucleobase” or simply a “base”). The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage. It is often preferable to include chemicalmodifications in oligonucleotides to alter their activity. Chemicalmodifications can alter oligonucleotide activity by, for example:increasing affinity of an antisense oligonucleotide for its target RNA,increasing nuclease resistance, and/or altering the pharmacokinetics ofthe oligonucleotide. The use of chemistries that increase the affinityof an oligonucleotide for its target can allow for the use of shorteroligonucleotide compounds.

The term “nucleobase” or “heterocyclic base moiety” as used herein,refers to the heterocyclic base portion of a nucleoside. In general, anucleobase is any group that contains one or more atom or groups ofatoms capable of hydrogen bonding to a base of another nucleic acid. Inaddition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (I), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable tothe present invention. The terms modified nucleobase and nucleobasemimetic can overlap but generally a modified nucleobase refers to anucleobase that is fairly similar in structure to the parent nucleobase,such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp,whereas a nucleobase mimetic would include more complicated structures,such as for example a tricyclic phenoxazine nucleobase mimetic. Methodsfor preparation of the above noted modified nucleobases are well knownto those skilled in the art.

Antisense compounds of the present invention may also contain one ormore nucleosides having modified sugar moieties. The furanosyl sugarring of a nucleoside can be modified in a number of ways including, butnot limited to, addition of a substituent group, bridging of twonon-geminal ring atoms to form a bicyclic nucleic acid (BNA) andsubstitution of an atom or group such as —S—, —N(R)— or —C(R₁)(R₂) forthe ring oxygen at the 4′-position. Modified sugar moieties are wellknown and can be used to alter, typically increase, the affinity of theantisense compound for its target and/or increase nuclease resistance. Arepresentative list of preferred modified sugars includes but is notlimited to bicyclic modified sugars (BNA's), including LNA and ENA(4′-(CH₂)₂—O-2′ bridge); and substituted sugars, especially2′-substituted sugars having a 2′-F, 2′-OCH₂ or a 2′-O(CH₂)₂—OCH₃substituent group. Sugars can also be replaced with sugar mimetic groupsamong others. Methods for the preparations of modified sugars are wellknown to those skilled in the art.

The present invention includes internucleoside linking groups that linkthe nucleosides or otherwise modified monomer units together therebyforming an antisense compound. The two main classes of internucleosidelinking groups are defined by the presence or absence of a phosphorusatom. Representative phosphorus containing internucleoside linkagesinclude, but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates.Representative non-phosphorus containing internucleoside linking groupsinclude, but are not limited to, methylenemethylimino(—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate(—O—C(O)(NH)—S—); siloxane (—O—Si(H)₂—O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Antisense compounds having non-phosphorusinternucleoside linking groups are referred to as oligonucleosides.Modified internucleoside linkages, compared to natural phosphodiesterlinkages, can be used to alter, typically increase, nuclease resistanceof the antisense compound. Internucleoside linkages having a chiral atomcan be prepared racemic, chiral, or as a mixture. Representative chiralinternucleoside linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

As used herein the term “mimetic” refers to groups that are substitutedfor a sugar, a nucleobase, and/or internucleoside linkage. Generally, amimetic is used in place of the sugar or sugar-internucleoside linkagecombination, and the nucleobase is maintained for hybridization to aselected target. Representative examples of a sugar mimetic include, butare not limited to, cyclohexenyl or morpholino. Representative examplesof a mimetic for a sugar-internucleoside linkage combination include,but are not limited to, peptide nucleic acids (PNA) and morpholinogroups linked by uncharged achiral linkages. In some instances a mimeticis used in place of the nucleobase. Representative nucleobase mimeticsare well known in the art and include, but are not limited to, tricyclicphenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods ofsynthesis of sugar, nucleoside and nucleobase mimetics are well known tothose skilled in the art.

As used herein the term “nucleoside” includes, nucleosides, abasicnucleosides, modified nucleosides, and nucleosides having mimetic basesand/or sugar groups.

In the context of this invention, the term “oligonucleotide” refers toan oligomeric compound which is an oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA). This term includesoligonucleotides composed of naturally- and non-naturally-occurringnucleobases, sugars and covalent internucleoside linkages, possiblyfurther including non-nucleic acid conjugates.

The present invention provides compounds having reactive phosphorusgroups useful for forming internucleoside linkages including for examplephosphodiester and phosphorothioate internucleoside linkages. Methods ofpreparation and/or purification of precursors or antisense compounds ofthe instant invention are not a limitation of the compositions ormethods of the invention. Methods for synthesis and purification of DNA,RNA, and the antisense compounds of the instant invention are well knownto those skilled in the art.

As used herein the term “chimeric antisense compound” refers to anantisense compound, having at least one sugar, nucleobase and/orinternucleoside linkage that is differentiallv modified as compared tothe other sugars, nucleobases and internucleoside linkages within thesame oligomeric compound. The remainder of the sugars, nucleobases andinternucleoside linkages can be independently modified or unmodified. Ingeneral a chimeric oligomeric compound will have modified nucleosidesthat can be in isolated positions or grouped together in regions thatwill define a particular motif Any combination of modifications and ormimetic groups can comprise a chimeric oligomeric compound of thepresent invention.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Certain chimeric as well as non-chimeric oligomeric compounds can befurther described as having a particular motif. As used in the presentinvention the term “motif” refers to the orientation of modified sugarmoieties and/or sugar mimetic groups in an antisense compound relativeto like or differentially modified or unmodified nucleosides. As used inthe present invention, the terms “sugars”, “sugar moieties” and “sugarmimetic groups” are used interchangeably. Such motifs include, but arenot limited to, gapped motifs, alternating motifs, fully modifiedmotifs, hemtimer motifs, blockmer motifs, and positionally modifiedmotifs. The sequence and the structure of the nucleobases and type ofinternucleoside linkage is not a factor in determining the motif of anantisense compound.

As used in the present invention the term “gapped motif” refers to anantisense compound comprising a contiguous sequence of nucleosides thatis divided into 3 regions, an internal region (gap) flanked by twoexternal regions (wings). The regions are differentiated from each otherat least by having differentially modified sugar groups that comprisethe nucleosides. In some embodiments, each modified region is uniformlymodified (e.g. the modified sugar groups in a given region areidentical); however, other motifs can be applied to regions. Forexample, the wings in a gapmer could have an alternating motif. Thenucleosides located in the gap of a gapped antisense compound have sugarmoieties that are different than the modified sugar moieties in each ofthe wings. In a preferred embodiment of the invention, the antisensecompounds are 5-10-5 MOE gapmers having a 2′-MOE modifications onnucleobases 1-5 and 16-20, all cytosines are 5MeC, and a fullphosphorothioate backbone.

As used in the present invention the term “alternating motif” refers toan antisense compound comprising a contiguous sequence of nucleosidescomprising two differentially sugar modified nucleosides that alternatefor essentially the entire sequence of the antisense compound, or foressentially the entire sequence of a region of an antisense compound.

As used in the present invention the term “fully modified motif” refersto an antisense compound comprising a contiguous sequence of nucleosideswherein essentially each nucleoside is a sugar modified nucleosidehaving uniform modification.

As used in the present invention the term “hemimer motif” refers to asequence of nucleosides that have uniform sugar moieties (identicalsugars, modified or unmodified) and wherein one of the 5′-end or the3′-end has a sequence of from 2 to 12 nucleosides that are sugarmodified nucleosides that are different from the other nucleosides inthe hemimer modified antisense compound.

As used in the present invention the term “blockmer motif” refers to asequence of nucleosides that have uniform sugars (identical sugars,modified or unmodified) that is internally interrupted by a block ofsugar modified nucleosides that are uniformly modified and wherein themodification is different from the other nucleosides. Methods ofpreparation of chimeric oligonucleotide compounds are well known tothose skilled in the art.

As used in the present invention the term “positionally modified motif”comprises all other motifs. Methods of preparation of positionallymodified oligonucleotide compounds are well known to those skilled inthe art.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), a or B, or as (D) or (L) such as foramino acids et al. The present invention is meant to include all suchpossible isomers, as well as their racemic and optically pure forms.

In one aspect of the present invention antisense compounds are modifiedby covalent attachment of one or more conjugate groups. Conjugate groupsmay be attached by reversible or irreversible attachments. Conjugategroups may be attached directly to antisense compounds or by use of alinker. Linkers may be mono- or bifunctional linkers. Such attachmentmethods and linkers are well known to those skilled in the art. Ingeneral, conjugate groups are attached to antisense compounds to modifyone or more properties. Such considerations are well known to thoseskilled in the art.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides can be routinelyperformed according to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA: Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Antisense compounds of the present invention can be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives. The invention is not limited by the method of antisensecompound synthesis.

Oligomer Purification and Analysis

Methods of oligonucleotide purification and analysis are known to thoseskilled in the art. Analysis methods include capillary electrophoresis(CE) and electrospray-mass spectroscopy. Such synthesis and analysismethods can be performed in multi-well plates. The method of theinvention is not limited by the method of oligomer purification.

Salts, Prodrugs and Bioequivalents

The antisense compounds of the present invention comprise anypharmaceutically acceptable salts, esters, or salts of such esters, orany other functional chemical equivalent which, upon administration toan animal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, the disclosure is also drawn to prodrugs andpharmaceutically acceptable salts of the antisense compounds of thepresent invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes, chemicals, and/or conditions. In particular, prodrug versionsof the oligonucleotides of the invention are prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510 or WO 94/26764. Prodrugs can also includeantisense compounds wherein one or both ends comprise nucleobases thatare cleaved (e.g., by incorporating phosphodiester backbone linkages atthe ends) to produce the active compound.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Sodium salts of antisense oligonucleotides are useful and are wellaccepted for therapeutic administration to humans. In anotherembodiment, sodium salts of dsRNA compounds are also provided.

Formulations

The antisense compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. In a preferred embodiment,administration is topical to the surface of the respiratory tract,particularly pulmonary, e.g., by nebulization, inhalation, orinsufflation of powders or aerosols, by mouth and/or nose.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers,finely divided solid carriers, or both, and then, if necessary, shapingthe product (e.g., into a specific particle size for delivery). In apreferred embodiment, the pharmaceutical formulations of the instantinvention are prepared for pulmonary administration in an appropriatesolvent, e.g., water or normal saline, possibly in a sterileformulation, with carriers or other agents to allow for the formation ofdroplets of the desired diameter for delivery using inhalers, nasaldelivery devices, nebulizers, and other devices for pulmonary delivery.Alternatively, the pharmaceutical formulations of the instant inventionmay be formulated as dry powders for use in dry powder inhalers.

A “pharmaceutical carrier” or “excipient” can be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal andare known in the art. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition.

Combinations

Compositions of the invention can contain two or more antisensecompounds. In another related embodiment, compositions of the presentinvention can contain one or more antisense compounds, particularlyoligonucleotides, targeted to a first nucleic acid and one or moreadditional antisense compounds targeted to a second nucleic acid target.Alternatively, compositions of the present invention can contain two ormore antisense compounds targeted to different regions of the samenucleic acid target. Two or more combined compounds may be used togetheror sequentially. Compositions of the instant invention can also becombined with other non-antisense compound therapeutic agents.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the references, GenBank accession numbers, and the like recited inthe present application is incorporated herein by reference in itsentirety.

Example 1 Transfection Methods

Cell Types

The effect of antisense compounds on target nucleic acid expression wastested in the following cell types.

T-24 Cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5 A basal media (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells wereroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells were seeded into 96-well plates(Falcon-Primaria #3872) at a density of approximately 4000-6000cells/well for use in oligomeric compound transfection experiments

A549:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (Manassas, Va.). A549 cells were routinelycultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum, 100 units per mlpenicillin, and 100 micrograms per ml streptomycin (Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of approximately 5000 cells/well for use inantisense compound transfection experiments.

b.END:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Institute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached approximately90% confluence Cells were seeded into 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately3000 cells/well for use in oligomeric compound transfection experiments.

Treatment with Antisense Compounds

When cells reach appropriate confluency, they are treated witholigonucleotide using a transfection lipid and method, such asLipofectin™ essentially by the manufacturer's instructions, asdescribed.

Briefly, when cells reached 65-75% confluency, they were treated witholigonucleotide. Oligonucleotide was mixed with LIPOFECTIN™ InvitrogenLife Technologies, Carlsbad, Calif.) in Opti-MEMT™ reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture wasincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture was replaced with fresh culturemedium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

Other transfection reagents and methods (e.g., electroporation) fordelivery of oligonucleotides to the cell are well known. The method ofdelivery of oligonucleotide to the cells is not a limitation of theinstant invention.

Control Oligonucleotides

Control oligonucleotides are used to determine the optimal antisensecompound concentration for a particular cell line. Furthermore, whenantisense compounds of the invention are tested in antisense compoundscreening experiments or phenotypic assays, control oligonucleotides aretested in parallel with compounds of the invention.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. The concentration ofpositive control oligonucleotide that results in 80% inhibition of thetarget mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of the target mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM when theantisense oligonucleotide is transfected using a liposome reagent and 1μM to 40 μM when the antisense oligonucleotide is transfected byelectroporation.

Example 2 Real-time Quantitative PCR Analysis of STAT 6 mRNA Levels

Quantitation of STAT 6 mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured were evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. After isolation theRNA is subjected to sequential reverse transcriptase (RT) reaction andreal-time PCR, both of which are performed in the same well. RT and PCRreagents were obtained from Invitrogen Life Technologies (Carlsbad,Calif.). RT, real-time PCR was carried out in the same by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR were normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT,real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA was quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).

170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well platecontaining 30 μL purified cellular RNA. The plate was read in aCytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm andemission at 530 nm.

The GAPDH PCR probes have JOE covalently linked to the 5′ end and TAMRAor MGB covalently linked to the 3′ end, where JOE is the fluorescentreporter dye and TAMRA or MGB is the quencher dye. In some cell types,primers and probe designed to a GAPDH sequence from a different speciesare used to measure GAPDH expression. For example, a human GAPDH primerand probe set is used to measure GAPDH expression in monkey-derivedcells and cell lines.

Probes and primers for use in real-time PCR were designed to hybridizeto target-specific sequences. Design of probes and primers is well withthe ability of those skilled in the art. The target-specific PCR probeshave FAM covalently linked to the 5′ end and TAMRA or MGB covalentlylinked to the 3′ end, where FAM is the fluorescent dye and TAMRA or MGBis the quencher dye.

Example 3 Antisense Inhibition of Human STAT 6 Expression by AntisenseCompounds

A series of antisense compounds was designed to target different regionsof human STAT 6 RNA, using published sequences or portions of publishedsequences as cited in Table 1, specifically GenBank numberNM_(—)003153.3 (SEQ ID NO: 1). A number antisense compounds taught in USPatent Publications US20040115634 and 20050239124, can also be mapped toSEQ ID NO: 1 and are shown in Table 2. In the inhibition studies, T-24cells were treated with 100 nM of oligonicleotide using LIPOFECTIN™.Inhibition of mRNA target expression was determined using the RT-PCRmethod detailed above. The results are shown in Table 2. TABLE 2Inhibition of human STAT 6 mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET % SEQ IDISIS # SITE SEQUENCE INHIB NO 153765 2057 AGTGAGCGAATGGACAGGTC 96 18153766 2589 CGCTGTCACTGGCTGGCTCA 83 19 153767  127 TTGATGATTTCTCCAGTGCT92 20 153768 1046 AGGACTTCATCCAGCCGGCC 50 21 153769 1160CCCAGGAACCTCAAGCCCAA 68 22 153770  348 GTCACCCAGAAGATGCCGCA 53 23 1537712206 TTTCCACGGTCATCTTGATG 59 24 153772  485 AAGATGGTGCTCCCCTCCCC 34 25153773  871 GCCGTTTCCAAATCTGGATC 76 26 153774  835 CTTTGGCTGCCTCTAGCTCT89 27 153775 1831 GTTTGGTGAGGTCCAGGACA 89 28 153776 2427CATCTGCAGGTGAGGCTCCT 85 29 153777 2782 TGGCCCTTAGGTCCATGTGG 76 30 1537782011 CTATCTGTGGAGAGCCATCC 72 31 153779 1911 ATTGAGAAGAAGGCTAGTAA 83 32153780  498 GCTGATGTGTTGCAAGATGG 78 33 153781 3125 GCCCCATCACCCTCAGAGAG80 34 153782  523 CCCTCTGATATATGCTCTCA 73 35 153783 1903GAAGGCTAGTAACGTACTGT 84 36 153784 1925 GTTCCGTCGGGCTCATTGAG 92 37 1537852585 GTCACTGGCTGGCTCAGGCA 87 38 153786 1619 TTCAGAGTTTCACACATCTT 83 39153787  185 CAGGCCCCATAGGTCTGTAG 88 40 153788  750 TATCAAGCTGTGCAGAGACA80 41 153789  378 CAGGAACTCCCAGGGCTGGC 74 42 153790 3106GCTCTGTATGTGTGTGTGCG 90 43 153791 2078 AGATCCCGGATTCGGTCCCC 88 44 153792 900 CGGTGCGCCATTCCCTGCCA 94 45 153793 2103 GGGATAGAGATTTTTGAGCT 53 46153794  252 GATCTGGGACTTGGAGGTTG 71 47 153795 2271 TCCAAGGTCATAAGAAGGCA88 48 153796 1868 ATGATCAGCCGGTCAGACCA 84 49 153797 1311CCCAGGAATGCTGTTCTCCA 88 50 153798 1050 TCTCAGGACTTCATCCAGCC  6 51 1537991777 CCAGCAGGATCTCCTTGTTG 82 52 153800 1266 TCCAGTGCTTTCTGCTCCAG 87 53153801  434 ACAGTGTCTGAAAGTAGGGC 50 54 195427  145 GCTGGCCCTGCTAGCACCTC68 55 195428  264 CCACAGAGACATGATCTGGG 79  2 195429  647GTCTTAAACTTGAGTTCTTC 53 56 195430  824 TCTAGCTCTCCAGTGGTCTC 78 57 1954311191 GGCCCTGACCAGCGGAGGCT 72 58 195432 1396 CCTCTGTGACAGACTCAGTG 72 59195433 1721 TCCATACTGAGGCTGTTGTC 20 60 195434 1993 CCTGGCCCCGGATGACATGG53 61 195435 2258 GAAGGCACCATGGTAGGCAT 55 62 195436 2612CCAATCCAAGTGCCCTGAGG 70 63 195437 2805 CAGCTGGGATCACCAACTGG 49 64 1954383050 GTGTCTCAGAGCCTGAACTT 77 65 195439   14 TAAGCAGTGGCTGCCCCAGC 51 66195440   29 CCTCCCTCTTCAGTGTAAGC 65 67 195441 3177 AGAAGCCTTCCATGCCCTAA83 68 195442 3222 TATGTTCCTGCCTATCCGTC 76 69 195443 3523CAACTAAGGTGCCAGCTATA 86 70 195444 3531 TGGTCATGCAACTAAGGTGC 84 71 1954453577 ATTTGTGTTGTCACGTAGGC 84 72 195446 3591 TCTCACCCTCCCAAATTTGT 48 73195447 3621 AGCACACTTGCTGCTGTCTT 74 74 195448 3771 GCCAGGCCTGGACCCAGACT60 75 195449 3827 GGGCAACAGAAAAGATGCAG 50 76 195453 1558CCATCTCAGAGAAGGCATTG 81 77

A series of antisense compounds was designed to target different regionsof human STAT 6 RNA, using published sequences or portions of publishedsequences as cited in Table 1, specifically GenBank numberNM_(—)003153.3 (SEQ ID NO: 1). In the inhibition studies, A549 cellswere treated with 50 nM of oligonucleotide using LIPOFECTIN™. Inhibitionof in RNA target expression was determined using the RT-PCR methoddetailed above. The results are shown in Tables 3 and 4. TABLE 3Inhibition of human STAT 6 mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ ID ISIS# SITE SEQUENCE (5′ to 3′) % INHIB NO 369370 12 AGCAGTGGCTGCCCCAGCCC 5978 369371 20 TCAGTGTAAGCAGTGGCTGC 66 79 369372 27 TCCCTCTTCAGTGTAAGCAG66 80 369373 154 GAGTTCAAGGCTGGCCCTGC 75 81 369374 248TGGGACTTGGAGGTTGCCTC 52 82 369375 253 TGATCTGGGACTTGGAGGTT 64 83 369376258 AGACATGATCTGGGACTTGG 80 84 369377 263 CACAGAGACATGATCTGGGA 70 85369378 268 GACCCCACAGAGACATGATC 29 86 369379 272 ACCAGACCCCACAGAGACAT 3587 369380 279 CTTGGAGACCAGACCCCACA 48 88 369381 284 GGCATCTTGGAGACCAGACC69 89 369382 351 CCAGTCACCCAGAAGATGCC 14 90 369383 356TCCAGCCAGTCACCCAGAAG 37 91 369384 426 TGAAAGTAGGGCACTAGCCA 51 92 369385431 GTGTCTGAAAGTAGGGCACT 77 93 369386 439 GCTGGACAGTGTCTGAAAGT 72 94369387 497 CTGATGTGTTGCAAGATGGT 74 95 369388 525 GTCCCTCTGATATATGCTCT 7696 369389 546 AGTGGCCACCAGCTTCAGGG 51 97 369390 551 CTGAAAGTGGCCACCAGCTT64 98 369391 561 AAGTATTTGTCTGAAAGTGG 74 99 369392 567TCCTTGAAGTATTTGTCTGA 48 100 369393 615 GAAAGGCATTGGCAAGTGGC 81 101369394 620 CAGTGGAAAGGCATTGGCAA 80 102 369395 630 TTCCTGCTTCCAGTGGAAAG70 103 369396 641 AACTTGAGTTCTTCCTGCTT 75 104 369397 740TGCAGAGACACTTGGCCAGC 53 105 369398 760 CAGGAGTTTCTATCAAGCTG 77 106369399 765 ATTAGCAGGAGTTTCTATCA 25 107 369400 770 GTCCCATTAGCAGGAGTTTC70 108 369401 775 GCCCAGTCCCATTACCAGGA 75 109 369402 780ACTTGGCCCAGTCCCATTAG 44 110 369403 786 GGCCTCACTTGGCCCAGTCC 55 111369404 792 GGCCAGGGCCTCACTTGGCC 38 112 369405 857 TGGATCCTCTTCAGCACTAG32 113 369406 862 AAATCTGGATCCTCTTCAGC 45 114 369407 868GTTTCCAAATCTGGATCCTC 66 115 369408 959 GAATAAATGTCCACCAGGCT 35 116369409 968 TGTAGCTGGGAATAAATGTC 69 117 369410 1121 TGGAACTTGGTCTGAGTCTT80 118 369411 1128 TCCAGCCTGGAACTTGGTCT 83 119 369412 1152CCTCAAGCCCAACAGGAATC 56 120 369413 1238 CCCTGAGGCACACTCAGCTC 74 121369414 1243 CAGGACCCTGAGGCACACTC 72 122 369415 1250 CCAGCCCCAGGACCCTGAGG75 123 369416 1286 ACAGTGTTGTTGATGATTTC 69 124 369417 1295TCCAAGGGCACAGTGTTGTT 87 125 369418 1318 AGCAGTTCCCAGGAATGCTG 77 126369419 1323 AGAGCAGCAGTTCCCAGGAA 68 127 369420 1328 AGGGCAGAGCAGCAGTTCCC65 128 369421 1338 GTTCTTGAACAGGGCAGAGC 62 129 369422 1348TGAGAAGCAGGTTCTTGAAC 55 130 369423 1353 CTTCTTGAGAAGCAGGTTCT 62 131369424 1360 GCTTGATCTTCTTGAGAAGC 68 132 369425 1392 TGTGACAGACTCAGTGCCCT84 133 369426 1424 CTGGCAGAGAAGAGCACAGC 63 134 369427 1429TGAAGCTGGCAGAGAAGAGC 59 135 369428 1439 GGGCCAAGTGTGAAGCTGGC 73 136369429 1471 ACAGGGCCTGGAGCTGGATG 40 137 369430 1477 GCAGAGACAGGGCCTGGAGC59 138 369431 1585 GCTCAGCCACCACAAAGGGC 73 139 369432 1620GTTCAGAGTTTCACACATCT 81 140 369433 1641 CACCTCAGCCATGAACTTCA 29 141369434 1646 GTCCCCACCTCAGCCATGAA 35 142 369435 1651 GGTTGGTCCCCACCTCAGCC83 143 369436 1672 AGTGCTCTGGGAGCAGCCCC 76 144 369437 1677GAGGAAGTGCTCTGGGAGCA 67 145 369438 1686 GGCCAGGAAGAGGAAGTGCT 55 146369439 1694 ATCTTCTGGGCCAGGAAGAG 11 147 369440 1699 TGAAGATCTTCTGGGCCAGG47 148 369441 1704 GTCATTGAAGATCTTCTGGG 30 149 369442 1708TGTTGTCATTGAAGATCTTC 65 150

TABLE 4 Inhibition of human STAT 6 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET % SEQ ID ISIS # SITE SEQUENCE (5′ to 3′) INHIB NO 342160 1821GTCCAGGACACCATCAAACC 61 151 369452 1800 CTGCCAAAAGGTGAAGCCAC 64 152369453 1816 GGACACCATCAAACCACTGC 72 153 369454 2004 TGGAGAGCCATCCTGGCCCC53 154 369455 2024 GGCTGGATGTTCTCTATCTG 68 155 369456 2029AGAATGGCTGGATGTTCTCT 63 156 369457 2034 GGCAGAGAATGGCTGGATGT 86 157369458 2044 ACAGGTCTTTGGCAGAGAAT 70 158 369459 2050 GAATGGACAGGTCTTTGGCA64 159 369460 2091 TTTGAGCTGAGCAAGATCCC 64 160 369461 2121CTCATCCTTGGGCTTCTTGG 70 161 369462 2128 GGAAAGCCTCATCCTTGGGC 72 162369463 2149 GTTCAGGCTTGTAGTGGCTC 58 163 369464 2175 ATAACCCCTGCCATCCTTAC37 164 369465 2180 GGGACATAACCCCTGCCATC 79 165 369466 2190GATGGTAGCTGGGACATAAC 57 166 369467 2199 GGTCATCTTGATGGTAGCTG 54 167369468 2245 TAGGCATCTGGAGCTCTGGG 55 168 369469 2250 CATGGTAGGCATCTGGAGCT62 169 369470 2263 CATAAGAAGGCACCATGGTA 20 170 369471 2270CCAAGGTCATAAGAAGGCAC 76 171 369472 2278 GGGCCATTCCAAGGTCATAA 57 172369473 2296 TGCTCATGGAGGAATCAGGG 33 173 369474 2301 CTGCATGCTCATGGAGGAAT36 174 369475 2311 CTGGGCCAAGCTGCATGCTC 38 175 369476 2316CATATCTGGGCCAAGCTGCA 28 176 369477 2321 GGCACCATATCTGGGCCAAG 53 177369478 2341 AGTGTGGTGGGTACACCTGG 62 178 369479 2429 GGCATCTGCAGGTGAGGCTC73 179 369480 2454 CAGGCTCATCTGGCCCAGGC 71 180 369481 2522GGGCTGGACACAGCATGCTC 64 181 369482 2527 GGTCAGGGCTGGACACAGCA 74 182369483 2547 CACATCTGAGCAGAGCAGGG 68 183 369484 2557 CCACCATGGTCACATCTGAG83 184 369485 2563 TGTCTTCCACCATGGTCACA 67 185 369486 2584TCACTGGCTGGCTCAGCCAG 67 186 369487 2613 ACCAATCCAAGTGCCCTGAG 73 187369488 2618 TCTTCACCAATCCAAGTGCC 53 188 369489 2623 ATATGTCTTCACCAATCCAA68 189 369490 2633 AGAGGAGGGAATATGTCTTC 52 190 369491 2639GGCAGCAGAGGAGGGAATAT 47 191 369492 2669 AGAAGCTTAGTGAGGTCCTG 66 192369493 2748 AGATTGCCCATAGTGGGAGG 50 193 369494 2754 GATCCCAGATTGCCCATAGT62 194 369495 2759 ATTGAGATCCCAGATTGCCC 69 195 369496 2764GGGACATTGAGATCCCAGAT 65 196 369497 2774 AGGTCCATGTGGGACATTGA 43 197369498 2781 GGCCCTTAGGTCCATGTGGG 56 198 369499 2786 GGGTTGGCCCTTAGGTCCAT72 199 369500 2864 AAGTGTCCAGAGCAGGTCTG 87 200 369501 2894TCCCCATCTGCTGCTTGGCA 74 201 369502 3034 ACTTCCCTTCCAGTCAGTGC 76 202369503 3040 GCCTGAACTTCCCTTCCAGT 90 203 369504 3267 AGACCCAATATCCTCTATCC56 204 369505 3272 GGCTGAGACCCAATATCCTC 86 205 369506 3303GGGTCCCTTGAGCTGCTTCC 42 206 369507 3340 TAACCACATGTCCAGACCCC 67 207369508 3440 TACTTTTGCATAGTCTCATA 83 208 369509 3447 GCCCTTGTACTTTTGCATAG71 209 369510 3540 TGTTCTATGTGGTCATGCAA 82 210 369511 3545ACACATGTTCTATGTGGTCA 84 211 369513 3627 GAGGCCAGCACACTTGCTGC 53 212369515 3642 TTAGCATATGTCAGAGAGGC 86 213 369516 3684 CACTTGGGCACAGTCAGACT78 214 369518 3689 GGACCCACTTGGGCACAGTC 84 215 369520 3694CACTTGGACCCACTTGGGCA 75 216 369522 3704 ATGTCACAGCCACTTGGACC 71 217369523 3761 GACCCAGACTCTCACCCTGG 82 218 369524 3768 AGGCCTGGACCCAGACTCTC52 219 369525 3793 TCATACACTGGAGGGCCACA 62 220 369526 3899GCTTAGGATCTATGACCCCT 83 221

The screen identified active target segments within the human STAT 6mRNA sequence. Each active target segment was targeted by multiple,active antisense oligonucleotides. These regions include nucleotides615-658; 1121-1171; 1318-14112929-2967; 2522-2582; 3540-3564; and3761-3787 of SEQ ID NO: 1. All of the oligonucleotides tested withineach of these regions inhibited expression of human STAT 6 RNA at least50%, and over half of the oligonucleotides tested inhibited expressionat least 65%. The screen also identified inactive target segments,regions to which multiple inactive antisense oligonucleotides weretargeted. These regions include nucleotides 1641-1665 and 2296-2335 ofSEQ ID NO: 1. All of the oligonucleotides tested inhibited expression ofhuman STAT 6 RNA 38% or less. Identification of these regions allows forthe design of antisense oligonucleotides that modulate the expression ofSTAT 6.

Oligonucleotides targeted to the following sites on SEQ ID NO: 1 inhibitexpression of human STAT 6 RNA at least about 65% under the conditionsdescribed for the respective target sites above: 20-39, 27-46, 29-48,145-164, 154-173; 185-204; 252-271; 258-277, 263-282, 264-283, 284-303,378-397, 383-402, 431-450; 439-458, 497-516, 498-517, 523-542, 525-544,561-580, 615-634, 620-639, 630-649, 641-660, 968-987, 750-769, 760-779,770-789, 775-794, 824-843, 835-854, 868-887, 871-890, 900-919,1023-1042, 1121-1140, 1128-1147, 1160-1179, 1191-1210, 1238-1257,1243-1262, 1250-1269, 1266-1285, 1277-1296, 1286-1305, 1295-1314,1311-1330, 1318-1337, 1323-1342, 1328-1347, 1343-1362, 1360-1379,1392-1411, 1439-1458, 1558-1577, 1585-1604, 1607-1626, 1620-1639,1651-1670, 1672-1691, 1677-1696, 1708-1727, 1721-1740, 1777-1796,1816-1835, 1826-1845, 1831-1850, 1849-1868, 1868-1887, 1903-1922,1911-1930, 1925-1944, 2011-2030, 2022-2041, 2024-2043, 2034-2053,2044-2063, 2050-2069, 2053-2072, 2057-2076, 2063-2082, 2078-2097,2121-2140, 2128-2147, 2180-2199, 2270-2289, 2271-2290, 2427-2446,2429-2448, 2454-2473, 2527-2546, 2547-2566, 2557-2576, 2563-2582,2584-2603, 2585-2604, 2589-2608, 2612-2631, 2613-2632, 2623-2642,2656-2675, 2669-2688, 2759-2778, 2764-2783, 2782-2801, 2786-2805,2864-2883, 2894-2913, 3034-3053, 3040-3059, 3050-3069, 3106-3125,3125-3144, 3177-3196, 3222-3241, 3272-3291, 3340-3359, 3440-3459,3447-3466, 3523-3542, 3531-3550, 3540-3559, 3545-3564, 3577-3596,3621-3640, 3642-3661, 3684-3703, 3689-3708, 3694-3713, 3704-3723,3761-3780, 3889-3918. An active target segment can be bracketed by anyof the two segments listed above, provided that the requirements foractivity of the intervening oligonucleotides is met. Using the list andthe tables above, a subset of oligonucleotides with activity of at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,and at least about 90% can be readily identified. Such analyses are wellwithin the ability of those skilled in the art.

Example 4 Antisense Inhibition of Murine STAT 6 Expression by AntisenseCompounds

A series of antisense antisense compounds was designed to targetdifferent regions of mouse STAT 6 RNA, using published sequences citedin Table 1. In the instant screen, b.END cells were treated with 45 nMof oligonucleotide using LIPOFECTIN™. Inhibition of mRNA targetexpression was determined using the RT-PCR method detailed above. Fromthis screen, two of the oligonucleotides found to be active wereselected for further analysis, ISIS195428 (5′-CCACAGAGACATGATCTGGG-3′,SEQ ID NO: 2) and ISIS 342133 (5′-CCGACCAGGAACTCCCAGGG-3′, SEQ ID NO:3).

Both of the STAT 6 specific ASOs gave a dose dependent reduction in thetarget RNA. No significant change in RNA levels were observed with acontrol, non-specific ASO. This demonstrates that the STAT 6 ASOs areworking via a target specific mechanism.

Example 5 Mouse Models of Allergic Inflammation

Asthma is a complex disease with variations on disease severity andduration. In view of this, multiple animal models have been designed toreflect various aspects of the disease (see FIG. 1). It is understoodthat the models have some flexibility in regard to days of sensitizationand treatment, and that the timelines provided reflect the experimentalmethods used herein. There are several important features common tohuman asthma and the mouse model of allergic inflammation. One of theseis pulmonary inflammation, in which production of Th2 cytokines, e.g.,IL 4, IL 5, IL 9, and IL 13 is dominant. Another is goblet cellmetaplasia with increased mucus production. Lastly, airwayhyperresponsiveness (AHR) occurs, resulting in increased sensitivity tocholinergic receptor agonists such as acetylcholine or methacholine.

Ovalbumin Induced Allergic Inflammation—Acute Model

The acute model of induced allergic inflammation is a prophylaxistreatment paradigm. Animals are sensitized to allergen by systemicadministration (i.e., intraperitoneal injection), and treated with thetherapeutic agent prior to administration of the pulmonary allergenchallenge (see FIG. 1A). In this model, there is essentially nopulmonary inflammation prior to administration of the therapeutic agent.

Balb/c mice (Charles River Laboratory, Taconic Farms, N.Y.) weremaintained in micro-isolator cages housed in a specific pathogen free(SPF) facility. The sentinel cages within the animal colony surveyednegative for viral antibodies and the presence of known mouse pathogens.Mice were sensitized and challenged with aerosolized chicken OVA.Briefly, 20 ug of alum precipitated OVA was injected intraperitoneallyon days 0 and 14. On days 24, 25 and 26, the animals were exposed for 20minutes to 1% OVA (in saline) by ultrasonic nebulization using aLovelace nebulizer (Model 01-100). On days 17, 19, 21, 24, and 26animals were dosed intratracheally with 0.01 ug/kg, 0.1 ug/kg, 10 ug/kg,or 100 ug/kg of ISIS195428 or ISIS 342133 as well as a mismatch controloligonucleotide and/or vehicle control (0.9% normal saline). Analysiswas performed on day 28.

Effect of Pulmonary Administration of ASOs Targeted to STAT 6 on AirwayHyperreponsiveness in Response to Methacholine

Airway responsiveness was assessed by inducing airflow obstruction witha methacholine aerosol using a noninvasive method. This method usedunrestrained conscious mice that are placed into a test chamber of aplethsmograph (Buxco Electronics, Inc. Troy, N.Y.). Pressure differencebetween this chamber and a reference chamber were used extrapolateminute volume, breathing frequency and enhanced pause (Penh). Penh is adimensionless parameter that is a function of total pulmonary airflow inmice (i.e. the sum of the airflow in the upper and lower respiratorytracts) during the respiratory cycle of the animal. A lower Penh isindicative of greater the airflow. This parameter is known to closelycorrelate with lung resistance as measured by traditional, invasivetechniques using ventilated animals (see e.g., Hamelmann et al., 1997).

ISIS195428 or ISIS 342133, but not the vehicle or mismatcholigonucleotide control, caused a significant (p<0.05 v. control)suppression in methacholine induced AHR at all doses in sensitized miceas measured by whole body plethysmography.

Effect of Pulmonary Administration of ASOs Targeted to STAT 6 onInflammatory Cell Infiltration

The effect of ISIS195428 and ISIS 342133 on inflammatory cell profiles,particularly eosinophils, was analyzed. Cell differentials wereperformed on bronchial alveolar lavage (BAL) fluid collected from lungsof the treated mice after injection of a lethal dose of ketamine.Treatment with ISIS195428 and ISIS 342133, but not the vehicle ormismatch oligonucleotide control, resulted in a trend towards a decreasein BAL eosinophil (eos) infiltration. These results suggest that anoligonucleotide targeted to STAT 6 decreased pulmonary inflammation bydecreasing cosinophil infiltration.

These data demonstrate that STAT 6 targeted antisense oligonucleotideapproach is efficacious in decreasing pulmonary inflammation and airwayhyperresponsiveness in a prophylaxis model, and that STAT 6 is anappropriate target for the prevention, amelioration, and/or treatment ofAHR and pulmonary inflammation, and diseases associated therewith.

Mouse Model of Allergic Inflammation—Rechallenge Model

The rechallenge model of induced allergic inflammation allows testing ofa pharmacologic approach in mice that have been previously sensitizedand then exposed to an aeroallergen. During the first set of localallergen challenges, the mice develop allergen-specific memory Tlymphocytes. Subsequent exposure to a second set of inhaled allergenchallenges produces an enhanced inflammatory response in the lung, asdemonstrated by increased levels of Th2 cytokines in lavage fluid. Therechallenge model of allergic inflammation includes a second series ofaerosolized administration of OVA on days 59 and 60 in addition to thetwo IP OVA administrations on days 0 and 14 and the nebulized OVAadministration of days 24, 25 and 26 of the acute model (see FIG. 1B).Using this model, oligonucleotide treatment occurs after the first setof local allergen challenges. This also allows for the evaluation of thetarget's role in a recall response, as opposed to an initial immuneresponse.

In the rechallenge model, mice were treated with ISIS 195428; and amismatch control oligonucleotide and/or a vehicle control (i.e., 0.9%normal saline) on days 59, 61, 63, 66, and 68 delivered by nose onlyinhalation. In one experiment, oligonucleotides were dosed at 10, 100,and 500 ug/kg. In another experiment, oligonucleotides were dosed at0.1, 1, and 10 ug/kg. A Lovelace nebulizer (Model 01-100) was used todeliver the oligonucleotide into an air flow rate of 1.0 liter perminute feeding into a total flow rate of 10 liters per minute. Theexposure chamber was equilibrated with an oligonucleotide aerosolsolution for 5 minutes before mice were placed in a restraint tubesattached to the chamber. Restrained mice were treated for a total of 10minutes. The study endpoints can include many of those used in the acutemodel: Penh response (i.e., AHR reduction), inflammatory cells in BAL,mucus accumulation, cytokine production, and lung histology. STAT 6 RNAand protein level reductions in pulmonary structural and inflammatorycells can also be evaluated.

A significant (p<0.05 v. control) reduction in Penh was observed at the0.1, 10, 100, and 500 ug/kg doses of ISIS195428. A significant (p<0.05v. control) reduction in eosinophils in BAL was observed at all fivedoses of ISIS 195428.

These data demonstrate that STAT 6 targeted antisense oligonucleotideapproach is efficacious in decreasing pulmonary inflammation and airwayhyperresponsiveness, and that STAT 6 is an appropriate target for theprevention, amelioration, and/or treatment of AHR, pulmonaryinflammation, and diseases associated therewith.

Mouse Model of Allergic Inflammation—Chronic Model

The chronic model of induced allergic inflammation uses a therapeutictreatment regimen, with ASO treatment initiated after the establishmentlocal pulmonary inflammation. The chronic model recapitulates some ofthe histological features of severe asthma in humans, including collagendeposition and lung tissue remodeling. The chronic OVA model produces amore severe disease than that observed in the acute or rechallengedmodel.

This model includes intranasal OVA administration on days 14, 27-29, 47,61, and 73-75, at a higher dose (500 ug) than in the acute and chronicmodels, in addition to the two OVA IP administrations on days 0 and 14(see FIG. 1C). ISIS195428 and a vehicle control were administered bynose-only aerosol at doses of 5 and 500 ug/kg on days 31, 38, 45, 52,59, 66, and 73. Analysis of endpoints was performed on day 76. BALinflammatory cells were also measured on day 62. Intranasaladministration of the allergen results in a higher dose of the allergendelivered to the lungs relative to delivery by nebulizer. The increasednumber of allergen challenges produces more severe inflammatory events,resulting in increased lung damage and pathology more reflective ofclinical asthma than other models, in the absence of therapeuticinterventions. Endpoints tested can include those in the acute andrechallenge model, such as Penh (AHR), BAL inflammatory cells andcytokines, lung histology, and mucus accumulation. A “lung inflammationscore” was also determined in this experiment. The score is acombination of a number of factors including PAS positive airways,inflammatory cell infiltrates, goblet cell hyperplasia, and otherindicators of inflammation. This model also allows for the analysis ofendpoints typically associated with chronic diseases, such as asthma andCOPD, including subepithelial fibrosis, collagen deposition, enhancedgoblet cell metaplasia and smooth muscle cell hyperplasia.

A significant (p<0.05 v. control) reduction in Penh was observed at boththe 5 and 500 ug/kg doses of ISIS195428. A significant (p<0.05 v.control) reduction in eosinophils and neutrophils in BAL was observed tothe 500 ug/kg dose on day 76. A significant (p<0.05 v. control)reduction in eosinophils was observed with both the 5 ug/kg and 500ug/kg doses on day 62. A significant (p<0.05 v. control) decrease inlung inflammation score was also observed in response to both doses ofISIS 195423.

These data demonstrate that STAT 6 targeted antisense oligonucleotideapproach is efficacious in decreasing pulmonary inflammation and airwayhyperresponsiveness, and that STAT 6 is an appropriate target for theprevention, amelioration, and/or treatment of AHR and pulmonaryinflammation, and diseases associated therewith.

Example 6 Mouse Model of Allergic Inflammation, Analysis for NasalRhinitis Endpoints

Mouse models of allergen—induced acute and chronic nasal inflammationsimilar to the allergic inflammation models above have been used tostudy allergic rhinitis in mice (Hussain et al., Larangyoscope. 112:1819-1826. 2002; Iwasaki et al., J. Allergy Clin Immunol. 112: 134-140.2003; Malm-Erjefaelt et al., Am J Respir Cell Mol. Biol.24:352-352.2001; McCusker et al., J Allergy Clin Immunol., 110: 891-898;Saito et el., Immunology. 104:226-234. 2001). In all of the models, themice are sensitized to OVA by injection, as above, followed byintranasal OVA instillation.

The most substantial difference in the models is in the endpointsanalyzed. Endpoints include, but are not limited to, the amount ofsneezing and nasal scratching immediately after administration ofallergen challenge (i.e. intranasal OVA), and nasal histology includingmucus and eosinophil counts and measurements of cytokines or otherinflammatory products in nasal lavage fluid or nasal tissues. Methodsfor performing such analyses are detailed in the references cited whichare incorporated herein by reference.

Example 7 Rodent Model of Smoking Induced Pulmonary Disease

Smoking is known to cause lung irritation and inflammation which canresult in a number of diseases in humans including, but not limited to,emphysema and COPD. A number of smoking animal models are well known tothose skilled in the art including those utilizing mice (Churg et al.,2002. Am. J. Respir. Cell. Mol. Biol. 27:368-347; Churg et al., 2004.Am. J. Respir. Crit. Care Med. 170:492-498, both incorporated herein byreference), rats (e.g., Sekhon et al., 1994. Am. J. Physiol.267:L557-L563, incorporated herein by reference), and guinea pigs(Selman et al., 1996. Am J. Physiol. 271:L734-L739, incorporated hereinby reference). Animals are exposed to whole smoke using a smokingapparatus (e.g., Sekhon et al., 1994. Am. J. Physiol. 267:L557-L563)well known to those skilled in the art.

Changes in lung physiology are correlated with dose and time ofexposure. In short term studies, cell proliferation and inflammationwere observed. In one study, exposure of rats to 7 cigarettes for 1, 2,or 7 days resulted in proliferation of pulmonary artery walls at thelevel of the membranous bronchioles (MB), respiratory bronchioles (RB),and alveolar ducts (AD). Endothelial cell proliferation was only presentin vessels associated with AD. In a separate study (Churg et al., 2002.Am. J. Respir. Cell. Mol. Biol. 27:368-347), mice exposed to whole smokefrom four cigarettes were shown to have an increase in neutrophils,desmosine (an indicator of elastin breakdown), and hydroxyproline (anindicator of collagen breakdown) after only 24 hours. In a long termstudy, an emphysema-like state was induced (Churg et al., 2004. Am. J.Respir. Crit. Care Med. 170:492-498). Mice exposed to whole smoke fromfour cigarettes using a standard smoking apparatus, for five days perweek for six months were found to have an increase in neutrophils andmacrophages in BALF as compared to control mice. Whole lung matrixmetalloproteinases (MMP)-2, -9, -12, and -13, and matrix type-1 (MT-1)proteins were increased. An increase in matrix breakdown products wasalso observed in BALF. These markers correlate with tissue destructionand are observed in human lungs with emphysema.

These models can be used to determine the efficacy of therapeuticinterventions for the prevention, amelioration, and/or treatment of thedamage and disease caused by cigarette smoke and/or other insults.Administration of oligonucleotide can be performed prior to, concurrentwith, and/or after exposure to smoke to provide a prophylactic ortherapeutic model. Both ISIS195428 and ISIS 342133 are 100%complimentary to both mouse and rat STAT 6; therefore, they can be usedin both mouse and rat studies. Dose ranges are determined by the time ofoligonucleotide administration relative to smoke inhalation, with lowerdoses (e.g., 1-100 ug/kg) required for prevention of lung damage. Higherdoses (e.g., 100-1000 ug/kg) are required for treatment after, oralternating with, smoke exposure. Positive control (e.g., smokeexposure, no oligonucleotide administration) and negative control (e.g.,no smoke exposure, with or without oligonucleotide treatment) animalsare also analyzed.

Endpoints for analysis include those discussed in the asthma modelsabove. Functional endpoints include AHR, resistance and compliance.Morphological changes include BAL cell, cytokine levels, histologicaldeterminations of alveolar destruction (i.e., increase in alveolarspace) and airway mucus accumulation, as well as tissue markers ofdisease including collagen and elastin. The emphysematous changesspecific to this model discussed in this example can also be analyzed todetermine the effect of the antisense oligonucleotide.

Example 8 Mouse Model of Elastase Induced Emphysema

Elastase is an essential mediator in lung damage and inflammationrelease by neutrophils recruited following smoke-induced damage. A ratmodel of emphysema has been developed to analyze the process of elastasemediated lung damage, and possible therapeutic interventions to prevent,ameliorate, and/or treat the pathologies associated with such damage andresulting disease (Kuraki et al., 2002. Am J Respir Crit Care Med.,166:496-500, incorporated herein by reference). Intratrachealapplication of elastase induced emphysematous changes in all lobes ofthe lung including severe lung hemorrhage as demonstrated by increasedhemoglobin in BALF; neutrophil accumulation in BALF; inhibition ofhyperinflation and degradation of elastic recoil. Histopathologicalchanges included elastase-induced airspace enlargement and breakdown ofalveoli. These changes are similar to those observed in human emphysema.

In the model, rats are treated with human sputum elastase (SE563,Elastin Products, Owensville, Mo.) without further purification. Ratsare treated with a sufficient dose of elastase, about 200 to 400 units,by intratracheal administration using a microsprayer. Alternatively,intratracheal administration can be performed as described above in themouse models. After sufficient time to allow for damage to occur, abouteight weeks, functional and morphological changes are analyzed. Asimilar model can be performed using mice with a lowered dose ofelastase relative to weight and/or lung area (e.g., 0.05 U of porcinepancreatic elastase/g body weight).

Administration of oligonucleotide can be performed prior to, concurrentwith, and/or after administration of elastase to provide a prophylacticor therapeutic model. Both ISIS195428 and ISIS 342133 are 100%complimentary to both mouse and rat STAT 6. Dose ranges are determinedby the time of oligonucleotide administration relative to elastaseadministration with lower doses (e.g., 1-100 ug/kg) required forprevention of lung damage. Higher doses (e.g., 100-1000 ug/kg) arerequired for treatment after, or alternating with, elastaseadministration. Positive control (e.g., elastase treatment, nooligonucleotide administration) and negative control (e.g., no elastase,with or without oligonucleotide treatment) animals are also analyzed.

Endpoints for analysis include those discussed in the asthma modelsabove. Functional endpoints include AHR, resistance and compliance.Morphological changes include BAL cell, cytokine levels, and mucusaccumulation. The emphysematous changes specific to this model discussedin this example can also be analyzed to determine the effect of theantisense oligonucleotide.

1-14. (canceled)
 15. A compound comprising a modified oligonucleotideconsisting of 12 to 30 linked nucleosides and having a nucleobasesequence comprising at least 12 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs: 2, 3, and 18 to
 221. 16. The compound of claim 15, consisting of asingle-stranded modified oligonucleotide.
 17. The compound of claim 16,wherein the nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO:
 1. 18. The compound of claim 16, wherein atleast one internucleoside linkage is a modified internucleoside linkage.19. The compound of claim 18, wherein each internucleoside linkage is aphosphorothioate internucleoside linkage.
 20. The compound of claim 16,wherein at least one nucleoside comprises a modified sugar.
 21. Thecompound of claim 20, wherein at least one modified sugar is a bicyclicsugar.
 22. The compound of claim 20, wherein at least one modified sugarcomprises a 2′-O-methoxyethyl.
 23. The compound of claim 16, wherein atleast one nucleoside comprises a modified nucleobase.
 24. The compoundof claim 23, wherein the modified nucleobase is a 5-methylcytosine. 25.The compound of claim 15, wherein the modified oligonucleotidecomprises: a gap segment consisting of linked deoxynucleosides; a 5′wing segment consisting of linked nucleosides; a 3′ wing segmentconsisting of linked nucleosides; wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment and wherein eachnucleoside of each wing segment comprises a modified sugar.
 26. Thecompound of claim 25, wherein the modified oligonucleotide comprises: agap segment consisting of ten linked deoxynucleosides; a 5′ wing segmentconsisting of five linked nucleosides; a 3′ wing segment consisting offive linked nucleosides; wherein the gap segment is positioned betweenthe 5′ wing segment and the 3′ wing segment, wherein each nucleoside ofeach wing segment comprises a 2′-O-methoxyethyl sugar; and wherein eachinternucleoside linkage is a phosphorothioate linkage.
 27. The compoundof claim 16, wherein the modified oligonucleotide consists of 20 linkednucleosides.
 28. A composition comprising a modified oligonucleotideconsisting of 12 to 30 linked nucleosides and having a nucleobasesequence comprising at least 12 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs: 1-100 or a salt thereof and a pharmaceutically acceptable carrieror diluent.
 29. The composition of claim 28, consisting of asingle-stranded oligonucleotide.
 30. The composition of claim 28,wherein the modified oligonucleotide consists of 20 linked nucleosides.31. A method comprising administering to an animal a compound comprisinga modified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 2, 3, and 18 to
 221. 32. The method ofclaim 31, wherein the animal is a human.
 33. The method of claim 32,wherein administering the compound treats airway hyperresponsiveness.34. The method of claim 32, wherein administering the compound treatspulmonary inflammation.
 35. The method of claim 32, comprisingco-administering the compound and a corticosteroid.
 36. The method ofclaim 35, wherein the compound and the corticosteroid are administeredconcomitantly.
 37. The method of claim 32, wherein administrationcomprises pulmonary administration.
 38. The method of claim 32, whereinadministration comprises aerosol administration.
 39. A method comprisingidentifying a human with asthma and administering to a human atherapeutically effective amount of a composition comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs: 2, 3, and 18 to
 221. 40. The method of claim 39, whereinthe treatment causes morphological changes to BAL cells.
 41. The methodof claim 39, wherein the treatment causes a decrease in cytokine levels.42. The method of claim 39, wherein the treatment reduces mucousproduction.
 43. The method of claim 39, comprising co-administering thecompound and a corticosteroid.
 44. A compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion of nucleobases615-658; 1121-1171; 1318-1411; 2929-2967; 2522-2582; 3540-3564; or3761-3787 of SEQ ID NO: 1, and wherein the nucleobase sequence of themodified oligonucleotide is at least 90% complementary to SEQ ID NO: 1.45. The compound of claim 44, wherein the modified oligonucleotide is atleast 95% complementary to SEQ ID NO:
 1. 46. The compound of claim 45,wherein the modified oligonucleotide is 100% complementary to SEQ IDNO:
 1. 47. The compound of claim 44, wherein the modifiedoligonucleotide hybridizes exclusively within nucleobases 615-658;1121-1171; 1318-1411; 2929-2967; 2522-2582; 3540-3564; or 3761-3787. 48.The compound of claim 45, wherein the modified oligonucleotidehybridizes exclusively within nucleobases 615-658; 1121-1171; 1318-1411;2929-2967; 2522-2582; 3540-3564; or 3761-3787.
 49. The compound of claim46, wherein the modified oligonucleotide hybridizes exclusively withinnucleobases 615-658; 1121-1171; 1318-1411; 2929-2967; 2522-2582;3540-3564; or 3761-3787.
 50. A compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising a portion of at least 8 contiguousnucleobases fully complementary to an equal length portion ofnucleobases 615-658; 1121-1171; 1318-1411; 2929-2967; 2522-2582;3540-3564; or 3761-3787 of SEQ ID NO: 1, and wherein the nucleobasesequence of the modified oligonucleotide is at least 90% complementaryto SEQ ID NO:
 1. 51. The compound of claim 50, wherein the modifiedoligonucleotide is at least 95% complementary to SEQ ID NO:
 1. 52. Thecompound of claim 51, wherein the modified oligonucleotide is 100%complementary to SEQ ID NO:
 1. 53. The compound of claim 50, wherein themodified oligonucleotide hybridizes exclusively within nucleobases615-658; 1121-1171; 1318-1411; 2929-2967; 2522-2582; 3540-3564; or3761-3787.
 54. The compound of claim 51, wherein the modifiedoligonucleotide hybridizes exclusively within nucleobases 615-658;1121-1171; 1318-1411; 2929-2967; 2522-2582; 3540-3564; or 3761-3787. 55.The compound of claim 52, wherein the modified oligonucleotidehybridizes exclusively within nucleobases 615-658; 1121-1171; 1318-1411;2929-2967; 2522-2582; 3540-3564; or 3761-3787.